Although electronic pacemakers represent standard of care for treatment of symptomatic bradyarrhythmias and heart failure, they have a number of limitations including no neurohumoral responsiveness, a relatively short battery life, potential for primary and secondary infections, electromagnetic inference from other devices, and no adaptation to growth of pediatric patients. This has stimulated development of novel gene and cell-based biopacemaker therapies with the goal to eventually supplement or replace the use of electronic pacemakers. Thus far, cell-based biopacemaker therapies have involved intramyocardial injection of spontaneously active embryonic stem cell derived cardiomyocytes or bone marrow derived stem cells genetically engineered to express pacemaking HCN gene. However, since the proliferation and differentiation of injected stem cells is difficult to control, stem cell-based biopacemakers may have heterogeneous and unstable phenotype, and potentially induce immune rejection, tumors or arrhythmias. Ideally, cell-based biological pacemaker therapies should involve the implantation of patient's own cells engineered to autonomously generate stable pacemaking rhythm. Therefore, we propose to develop a novel approach to cell-based biopacemaker therapy wherein tissue-engineered grafts made of terminally differentiated somatic cells with stable, genetically engineered pacemaking properties are used to normalize rhythm of diseased hearts. Specifically, in this exploratory proposal we will: 1) identify sets of genes that enable conversion of unexcitable cells into stable autonomous pacemakers and 2) study how geometry of engineered pacemaker tissues affects their ability to pace 2D and 3D cardiac networks in vitro. The knowledge obtained in this project will allow us to pursue in the future clinically relevant procedures for the successful application of tissue-engineered pacemaking patch in the treatment of heart rhythm abnormalities. PUBLIC HEALTH RELEVANCE: Use of electronic pacemakers to restore normal heart rhythm is associated with a number of limitations including high cost, bacterial infections, lead or battery failure, and inability to adapt pacing rate to physiological needs. Biological pacemakers hold potential to replace electronic pacemakers and resolve the above limitations. The goal of this exploratory proposal is to establish tissue and genetic engineering rules that would enable generation of stable autonomous biopacemaking tissues starting from electrically unexcitable cells.